Spirodela Oligorrhiza Research Articles

Analysis of the electronic structure of the primary electron donor of photosystem I of <i>Spirodela</i> <i>oligorrhiza</i> by photochemically induced dynamic nuclear polarization (photo-CIDNP) solid-state nuclear magnetic resonance (NMR)

Abstract. The electron donor in photosystem I (PSI), the chlorophyll dimer P700, is studied by photochemically induced dynamic nuclear polarization (photo-CIDNP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) on selectively 13C and uniformly 15N labeled PSI core preparations (PSI-100) obtained from the aquatic plant duckweed (Spirodela oligorrhiza). Light-induced signals originate from the isotope-labeled nuclei of the cofactors involved in the spin-correlated radical pair forming upon light excitation. Signals are assigned to the two donor cofactors (Chl a and Chl a') and the two acceptor cofactors (both Chl a). Light-induced signals originating from both donor and acceptor cofactors demonstrate that electron transfer occurs through both branches of cofactors in the pseudo-C2 symmetric reaction center (RC). The experimental results supported by quantum chemical calculations indicate that this functional symmetry occurs in PSI despite similarly sized chemical shift differences between the cofactors of PSI and the functionally asymmetric special pair donor of the bacterial RC of Rhodobacter sphaeroides. This contributes to converging evidence that local differences in time-averaged electronic ground-state properties, over the donor are of little importance for the functional symmetry breaking across photosynthetic RC species.

Photochemically induced dynamic nuclear polarization NMR on photosystem II: donor cofactor observed in entire plant

The solid-state photo-CIDNP (photochemically induced dynamic nuclear polarization) effect allows for increase of signal and sensitivity in magic-angle spinning (MAS) NMR experiments. The effect occurs in photosynthetic reaction centers (RC) proteins upon illumination and induction of cyclic electron transfer. Here we show that the strength of the effect allows for observation of the cofactors forming the spin-correlated radical pair (SCRP) in isolated proteins, in natural photosynthetic membranes as well as in entire plants. To this end, we measured entire selectively 13C isotope enriched duckweed plants (Spirodela oligorrhiza) directly in the MAS rotor. Comparison of 13C photo-CIDNP MAS NMR spectra of photosystem II (PS2) obtained from different levels of RC isolation, from entire plant to isolated RC complex, demonstrates the intactness of the photochemical machinery upon isolation. The SCRP in PS2 is structurally and functionally very similar in duckweed and spinach (Spinacia oleracea). The analysis of the photo-CIDNP MAS NMR spectra reveals a monomeric Chl a donor. There is an experimental evidence for matrix involvement, most likely due to the axial donor histidine, in the formation of the SCRP. Data do not suggest a chemical modification of C-131 carbonyl position of the donor cofactor.

Insights into the structural and physicochemical properties of small granular starches from two hydrophyte duckweeds, Spirodela oligorrhiza and Lemna minor

The structure and physicochemical properties of starches from two hydrophyte duckweeds, Spirodela oligorrhiza and Lemna minor, were investigated and compared in this study. The amylose content and average size of starches were determined to be 20.85%, 4.70 μm and 27.77%, 6.17 μm for Spirodela oligorrhiza and Lemna minor, respectively. The average chain length of two duckweed starches was measured to be around DP 28. The chain length distribution was observed to be greatly different from other reported starches for the high proportion of long chains (DP ≥ 37) over 50%. Wide-angle X-ray diffraction profiles of the two starch samples displayed typical B-type diffraction pattern. The gelatinization enthalpy-changes (ΔHgel) of two starch samples was about 10.40 J/g for two duckweed starches. The present results suggested the potential utilization of small granular starches from duckweed in functional foods and dietary supplement products.

ChemInform Abstract: Analysis of Electron Donors in Photosystems in Oxygenic Photosynthesis by Photo‐CIDNP MAS NMR

AbstractReview: 80 refs.

Molecular forms of phosphatase and ribonuclease in phosphate deficient and N,N-dimethylmorpholinium chloride treated Spirodela oligorrhiza (Lemnaceae)

Soluble, membrane bound, and extracellular phosphatases (EC 3.1.3.2 and 3.1.3.1) of control, N,N-dimethylmorphołinłum chloride (DMMC) treated, and phosphate deficient (-P) axenic <i>Spirodela oligorrhiza</i> plants were analysed by Sephadex G-150 gel filtration. Soluble, acid enzymes of control plants were separated into two molecular forms with apparent MW ≥ 400 000 and 85 000. Phosphatase with MW 34 000 replaced the latter isoenzyme in the presence of DMMC. Two alkaline enzymes with apparent MW 210 000 and 36 000 were detected in -P plants. Triton X-100 solubilized a number of acid and alkaline phosphatases from membrane material. DMMC caused the appearance of two membrane bound enzymes (MW 48 000 and 14 000) which were not detected in the control. Senescimg control and DMMC treated plants released an acid phosphatase (MW 48 000; pH optimum 5.2) into the nutrient medium. -P plants released, in addition ,an alkaline phosphatase (MW 170,000; pH optimum 7.8-8.2). Ribonucleases (EC 2.7.7.17.) with apparent MW 31000 and 28 000 daltons were induced by DMMC and -P, respectively.

Manipulating duckweed through genome duplication

Significant inter- and intraspecific genetic variation exists in duckweed, thus the potential for genome plasticity and manipulation is high. Polyploidy is recognised as a major mechanism of adaptation and speciation in plants. We produced several genome-duplicated lines of Landoltia punctata (Spirodela oligorrhiza) from both whole plants and regenerating explants using a colchicine-based cocktail. These lines stably maintained an enlarged frond and root morphology. DNA ploidy levels determined by florescence-activated cell sorting indicated genome duplication. Line A4 was analysed after 75 biomass doublings. Frond area, fresh and dry weights, rhizoid number and length were significantly increased versus wild type, while the growth rate was unchanged. This resulted in accumulation of biomass 17-20% faster in the A4 plants. We sought to determine if specific differences in gene products are found in the genome duplicated lines. Non-targeted ultra performance LC-quadrupole time of flight mass spectrometry was employed to compare some of the lines and the wild type to seek identification of up-regulated metabolites. We putatively identified differential metabolites in Line A65 as caffeoyl hexoses. The combination of directed genome duplication and metabolic profiling might offer a path for producing stable gene expression, leading to altered production of secondary metabolites.

The growth-retarding effect of daminozide in Spirodela oligorrhiza (Kurz) Hegelm., and its reversal by gibberellin

Butanedioic acid mono-(2,2 dimethylhydrazide) (daminozide), in a concentration of 10<sup>-3</sup> M retarded growth of <em>Spirodela oligorrhiza</em> and increased chlorophyll and protein content in the fronds. Gibberellin A<sub>3</sub> and benzylaminopurine partially alleviated the symptoms of daminozide action.

An evaluation of the physiological activity of 9-amine-9-fluorenephosphonic acid derivatives

The physiological activity of eleven 9-amine-9-fluorenephosphonic acid derivatives, synthesized at the Wrocław Polytechnic, was examined. The test plant was <i>Spirodela oligorrhiza</i>. The effect of these compounds on the increase of the dry matter of this plant was tested in eight-day experiments. The activity of the compounds was varied. The most toxic were nos. 2, 4, 9, 8, 5 and 6 which were lethal in low concentrations. Somewhat less toxic were nos. 7, 10 and 11; nos. 1 and 3 were the least toxic.

Preliminary estimation of physiological activity of 9-amine-9-fluorenephosphonic acid derivatives

The physiological activity of twenty one 9-amine-9-fluorenephosphonic acid derivatives was tested. The compounds were synthesized by the Wrocław Technical University. <i>Spirodela oligorrhiza</i> was used as the test plant. There was tested the inhibiting influence of these compounds on the growth of <i>Spirodela</i> and synergism with the herbicide "mecoprop". Ethyl esters substituted at the nitrogen atom with buthyl and heptyl group appeared to be most active. Their concentration of 0.5-1.5 × 10<sup>-5</sup> M inhibited the growth of <i>Spirodela</i> by 50%. Most of the 9-amine-9-fluorenephosphonic acid derivatives tested with different substitutes showed a strong synergism with the herbicide "mecoprop". An addition of these compounds to sublethal solution of the herbicide killed <i>Spirodela</i> within 10-20 houres.

Effects of Harvest Regime and Water Depth on Nutrient Recovery from Swine Wastewater by Growing Spirodela oligorrhiza

Harvest regime and water depth were investigated to determine their effects on nutrient recovery from swine wastewater by a Spirodela oligorrhiza system. The results show that harvesting less duckweed at shorter intervals favored nutrient removal and total biomass production. Harvesting 20% of duckweed twice a week led to 66.3 and 109.4% higher total nitrogen and total phosphorus removals, respectively, and a duckweed production of 20.0 g fresh biomass/m2 x d--39.6% higher than that of harvesting 80% of duckweed once every 4 weeks. The water depth of duckweed system was of great importance to total nutrient removal. At the water depth of 40 cm, total nitrogen and total phosphorus removals were 2.05 and 2.16 times higher, respectively, than those at the water depth of 10 cm. However, because of the larger amount of nutrients in a deeper system, it took a longer time for the nutrient concentrations to decrease.

Growing duckweed in swine wastewater for nutrient recovery and biomass production

Spirodela oligorrhiza, a promising duckweed identified in previous studies, was examined under different cropping conditions for nutrient recovery from swine wastewater and biomass production. To prevent algae bloom during the start-up of a duckweed system, inoculating 60% of the water surface with duckweed fronds was required. In the growing season, the duckweed system was capable of removing 83.7% and 89.4% of total nitrogen (TN) and total phosphorus (TP) respectively from 6% swine lagoon water in eight weeks at a harvest frequency of twice a week. The total biomass harvested was 5.30 times that of the starting amount. In winter, nutrients could still be substantially removed in spite of the limited duckweed growth, which was probably attributed to the improved protein accumulation of duckweed plants and the nutrient uptake by the attached biofilm (algae and bacteria) on duckweed and walls of the system.

High expression of transgene protein in Spirodela

The monocot family Lemnaceae (duckweed) is composed of small, edible, aquatic plants. Spirodela oligorrhiza SP is a duckweed with a biomass doubling time of about 2 days under controlled, axenic conditions. Stably transformed Spirodela plants were obtained following co-cultivation of regenerative calli with Agrobacterium tumefaciens. GFP activity was successfully monitored in different subcellular compartments of the plant and correlated with different targeting sequences. Transgenic lines were followed for a period of at least 18 months and more than 180 vegetative doublings (generations). The lines are stable in morphology, growth rate, transgene expression, and activity as measured by DNA-DNA and immunoblot hybridizations, fluorescence activity measurements, and antibiotic resistance. The level of transgene expression is a function of leader sequences rather than transgene copy number. A stable, transgenic, GFP expression level >25% of total soluble protein is demonstrated for the S. oligorrhiza system, making it among the higher expressing systems for nuclear transformation in a higher plant.

Radical Scavengers Suppress Low Frequency EMF Enhanced Proliferation in Cultured Cells and Stress Effects in Higher Plants

In previous publications, we reported that sinusoidal varying magnetic fields (SVMF) modify the activity and dynamics of the malignancy marker adenosine deaminase, and enhance the proliferation of chick embryo fibroblasts (CEF). While the SVMF examined by us (50, 60 & 100 Hz / 0.06–0.7 mT) were all below kT, they may have the potential of altering chemical processes in which excited radicals are involved. We tested this hypothesis in two experimental systems: CEF in culture and Spirodela oligorrhiza (Lemnaceae) (a small aquatic plant, commonly known as Duckweed). CEF were exposed to SVMF of 100 Hz/0.7 mT for 24 h. The addition of the exogenous radical scavengers catalase, superoxide dismutase or vitamin E to the cells during exposure significantly suppressed enhancement of cell proliferation caused by the field (by 79, 67 and 82%, respectively, as evaluated by the MTT colorimetric assay). 15N NMR analysis of Duckweed plants fed by 15N-labeled ammonium chloride and exposed to SVMF at 60 and 100 Hz/0.7 mT for 24 h, revealed augmented alanine production. Alanine did not accumulate in the absence of SVMF. The addition of vitamin C, a radical scavenger, reduced alanine production by 82%. Exposure to SVMF resulted in specific metabolic stress effects in Duckweed plants and enhanced proliferation of CEF. In both cases, it is suggested that free radicals are involved.

Purification and Partial Characterization of an Acid Phosphatase from Spirodela oligorrhiza and Its Affinity for Selected Organophosphate Pesticides

An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fast protein liquid chromatography and partially characterized. The enzyme was purified 1871-fold with a total yield of 40%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the pure acid phosphatase resolved a single protein band that migrated to approximately 60 kDa. Nondenaturing SDS-PAGE electrophoresis revealed a single protein band around 120 kDa after staining with Coomassie Brilliant blue. Quantitative gel filtration chromatography estimated a native molecular mass of this enzyme to be 120 kDa. Thus, this acid phosphatase likely functions as a homodimer, consisting of two similar 60 kDa subunits. An electrophoretic technique using the flourogenic substrate 4-methylumbelliferyl phosphate enabled visualization of an acid phosphatase activity that corresponded to the protein band at 120 kDa on a nondenaturing PAGE gel. It was determined that the acid phosphatase had a pH optimum of 6.0 at 25 degrees C. The enzyme activity appeared to be stable over a broad range of temperatures (10-40 degrees C) and in the presence of the metals Zn2+, Mn2+, and Mg2+ as well as the chelating agents ethylenedinitrilotetraacetic acid and ethylene glycol tetraacetic acid. It was shown that this acid phosphatase could hydrolyze a variety of physiological organophosphate compounds including beta-glycerophosphate, phosphoserine, adenosine triphosphate, adenosine diphosphate, adenosine monphosphate, and pyrophosphate. Furthermore, analysis using capillary electrophoresis demonstrated that this hydrolytic enzyme could transform a wide array of organophosphate pesticides including S-2-ethylthioethyl O,O-dimethylphosphorothioate (demeton-S-methyl); S-1,2-bis(ethoxycarbonyl)ethyl O,O-dimethylphosphorodithioate (malathion); O,O-dimethyl O-4-nitrophenyl (paraoxon); O,O,O,O-tetraethyldithiopyrophosphate (sulfatep); O-2-chloro-4-nitrophenyl O,O-dimethylphosphorothioate (dicapthon); and 2,2-dichlorovinyl dimethylphosphate (dichlorvos).

Induction of high affinity phosphate transporter in the duckweed Spirodela oligorrhiza.

Duckweed plants (Spirodela oligorrhiza) grown under phosphate (Pi)-deficient conditions (- P plants) exhibited more than 50-fold higher Pi uptake activity than plants grown under Pi-sufficient conditions (+ P plants). The Pi uptake activity of - P plants measured using (32)Pi was significantly inhibited by carbonylcyanide m-chlorophenylhydrazone, indicating that Pi uptake is energized by the electrochemical proton gradient across the plasma membrane (PM). When Pi uptake was examined at various concentrations of Pi, more active uptake of Pi was observed in - P plants than in + P plants, irrespective of the Pi concentrations. An immunoblot analysis of the PM proteins using antiserum against the conserved sequence of the high-affinity Pi transporter recognized the occurrence and large accumulation of a novel protein band at 48 kDa in - P plants. The protein was almost completely extracted with chloroform-methanol (2:1, v/v), but only a trace amount of the protein was detected in + P plants. Immunohistochemical studies of plant roots using the same antiserum demonstrated a large accumulation of high-affinity Pi transporters at the outermost cortical cells of - P plants, but not of + P plants. When an immunoblot analysis of PM proteins was performed using antiserum against the PM H(+)-ATPase, a positive band of about 96 kDa was detected in both plants with a similar signal intensity. Furthermore, ATP-hydrolytic and ATP-dependent H(+)-transporting activities of PM H(+)-ATPase in - P plants were not higher than those in + P plants. However, kinetic analyses showed that the PM H(+)-ATPase in - P plants had a lower K(m) value and a higher coupling efficiency between ATP hydrolysis and H(+) pumping than the corresponding values in + P plants. These results suggest that the significant stimulation of Pi uptake in - P plants may be due mainly to the induction and accumulation of the high-affinity Pi transporter in the PM, and that the electrochemical proton gradient across the PM may be generated by the high-ATP-affinity and energy-efficient H(+) pump in - P plants. This would facilitate the acquisition of Pi in S. oligorrhiza under Pi-depleted conditions.

Callus induction and regeneration in Spirodela and Lemna.

The development of tissue culture systems in duckweeds has, to date, been limited to species of the genus Lemna. We report here the establishment of an efficient tissue culture cycle (callus induction, callus growth and plant regeneration) for Spirodela oligorrhiza Hegelm SP, Spirodela punctata 8717 and Lemna gibba var. Hurfeish. Significant differences were found among the three duckweed species pertaining to carbohydrate and phytohormone requirements for callus induction, callus growth and frond regeneration. In vitro incubation with poorly assimilated carbohydrates such as galactose ( S. oligorrhiza SP and L. gibba var. Hurfeish) and sorbitol ( S. punctata 8717) as sole carbon source yielded high levels of callus induction on phytohormone-supplemented medium. Sorbitol is required for optimal callus growth of S. oligorrhiza SP and S. punctata 8717, while sucrose is required for callus growth of L. gibba var. Hurfeish. Sucrose either alone ( S. oligorrhiza SP, L. gibba var. Hurfeish) or in addition to sorbitol ( S. punctata 8717) is required for frond regeneration.

Probing the electronic structure of tyrosine radical YD in photosystem II by EPR spectroscopy using site specific isotope labelling in Spirodela oligorrhiza

Tyrosine (Y D ) in the D2 reaction centre polypeptide of photosystem II (PSII) is redox-active and, under illumination, forms a dark-stable radical Y D . The origin of its stability and the functional role of Y D are not well understood. For understanding the electronic structure and reactivity of Y D , it is crucial to unambiguosly establish its hyperfine structure. There is considerable variation in the hyperfine data of Y D and their interpretation in literature. In the present study, the hyperfine structure of tyrosine radical Y D in PSII was probed by EPR in conjunction with carefully designed site specific isotope labelling. A comprehensive series of different selectively 2 H-, 13 C- or 17 O-labeled tyrosine were synthesized and incorporated in Spirodela oligorrhiza with more than 95% enrichment. The 13 C- and 17 O-hyperfine interactions were obtained from spectral simulations. From the anisotropy of the hyperfine interactions the spin densities at all phenoxyl ring positions were precisely obtained. Comparison of the absolute differences in individual spin densities between Y D and neutral tyrosine radical in vitro with those of computationally calculated spin densities yield excellent agreement for a well ordered hydrogen bond between Y D and the surrounding protein matrix with a bond length of 1.5 Å. Enantioselective labeling confirms that the β-methylene hydrogens of Y D in S. oligorrhiza are oriented in a highly constrained specific position making Y D strongly immobilized, thereby ensuring a firm hydrogen bond of the phenoxyl oxygen to the protein matrix.

Phosphorylation of the D1 photosystem II reaction center protein is controlled by an endogenous circadian rhythm.

The light dependence of D1 phosphorylation is unique to higher plants, being constitutive in cyanobacteria and algae. In a photoautotrophic higher plant, Spirodela oligorrhiza, grown in greenhouse conditions under natural diurnal cycles of solar irradiation, the ratio of phosphorylated versus total D1 protein (D1-P index: [D1-P]/[D1] + [D1-P]) of photosystem II is shown to undergo reproducible diurnal oscillation. These oscillations were clearly out of phase with the period of maximum in light intensity. The timing of the D1-P index maximum was not affected by changes in temperature, the amount of D1 kinase activity present in the thylakoid membranes, the rate of D1 protein synthesis, or photoinhibition. However, when the dark period in a normal diurnal cycle was cut short artificially by transferring plants to continuous light conditions, the D1-P index timing shifted and reached a maximum within 4 to 5 h of light illumination. The resultant diurnal oscillation persisted for at least two cycles in continuous light, suggesting that the rhythm is endogenous (circadian) and is entrained by an external signal.

The influence of microcystin-LR and hepatotoxic cyanobacterial extract on the water plant Spirodela oligorrhiza.

The eutrophication of the Sulejów Reservoir dam in Poland is related to toxicity from cyanobacterial blooms. The main species responsible for hepatotoxic bloom formation is Microcystis aeruginosa. The aim of this study was to evaluate the influence of toxic cyanobacterial extract on the growth and morphology of the water plant Spirodela oligorrhiza, compared with commercial-grade microcystin-LR (MC-LR). It was found that after 96 h of incubation the highest concentration of cyanobacterial extract, containing 0.344 mg MC-LR/L, reduced the number of fronds by about 50% in comparison with the control. The extract effected a reduction in the frond mass and a decrease in chlorophyll (a + b) concentration. A reduction in the number of fronds was also observed after the first 24 h of incubation in the presence of 0.2 and 0.1 microg/L of commercial-grade MC-LR. Changes in activity of constitutive acid phosphatase and RNase after 7 days of incubation with commercial-grade MC-LR were observed. The results confirm the toxicity of cyanobacterial hepatotoxins to Spirodela oligorrhiza, which can be used as a sensitive bioindicator.

Cloning and characterization of cDNA of the GPI-anchored purple acid phosphatase and its root tissue distribution in Spirodela oligorrhiza.

A cDNA clone of the glycosylphosphatidylinositol (GPI)-anchored purple acid phosphatase (PAP) has been obtained by a combination of cDNA library screening and 5' rapid amplification of cDNA ends from Spirodela oligorrhiza plants grown under phosphate-deficient (-P) conditions. The open reading frame of the S. oligorrhiza PAP cDNA consists of 1 365 bp encoding a 455 amino acid protein. Its deduced amino acid sequence shows 82 and 80% similarity to Arabidopsis thaliana and Phaseolus vulgaris PAP, respectively. The amino acid residue, Ala439, followed by two more small amino acid residues, Asp and Ser, is predicted to be the GPI-anchoring (omega) site. The absence of a dibasic motif upstream of the putative omega site suggests that the PAP is a cell wall protein. This presumption is supported by the finding that PAP was released by digestion of the cell wall fraction with cellulase. The GPI anchor is speculated to be a signal for transporting PAP to the cell wall. Immunohistochemical results using -P plant roots demonstrate that PAP is preferentially distributed in the outermost cortical cells of roots but not in the epidermis, suggesting its role in acquiring inorganic phosphate under phosphate-deficient conditions. Northern blot analysis using the S. oligorrhiza PAP cDNA as a probe demonstrates that expression of the PAP gene increased during growth of -P plants and this time-dependent occurrence in mRNA levels of the PAP in -P plants was also observed in their protein and activity levels.